사찰의 된장에서 분리된 Bacillus Licheniformis YB-1234의 내열성 α-Amyalse

이은지 ( Eun Ji Lee ),윤기홍 ( Ki Hong Yoon ) 한국미생물생명공학회(구 한국산업미생물학회) 2012 한국미생물·생명공학회지 Vol.40 No.4

국내 사찰에서 제조된 된장으로부터 내열성 α-amylase 생산균으로 분리된 YB-1234는 형태적 특성, 생화학적 성질 및 16S rRNA 유전자 염기서열에 근거하여 Bacillus licheniformis로 동정되었다. B. licheniformis YB-1234의 α-amylase 유전자를 클로닝하여 그 염기서열을 결정하였으며 그로부터 유추된 α-amylase의 아미노산 서열은 glycosyl hydrolase family 13에 속하는 B. licheniformis의 내열성 α-amylases와 매우 높은 상동성을 보였다. α-Aamylase 유전자를 함유한 재조합 대장균과 B. licheniformis에 의해 각각 생산된 α-amylase는 pH 6.0에서 최대활성을 보였으나, 최적 반응온도는 약간의 차이가 있었다. 또한 B. licheniformis로부터 α-amylase는 재조합 대장균에서 생산된 효소보다 열안정성이 매우 높았다. 이들 효소에 의한 maltotetraose와 maltohexaose의 주된 가수분해산물로는 glucose, maltose 및 maltotriose가 관찰되었다. A bacterial strain was isolated from soybean paste fermented in a Korean Buddhist temple as a producer of the extracellular thermostable α-amylase. The isolate YB-1234 has been identified as Bacillus licheniformis on the basis of its 16S rDNA sequence, morphology and biochemical properties. A gene encoding the thermostable α-amylase of B. licheniformis YB-1234 was cloned into Escherichia coli and its nucleotide sequence was determined. The deduced amino acid sequence of α-amylase was very highly homologous to those of the thermostable α-amylases of B. licheniformis belonging to the glycosyl hydrolase family 13. The α-amylase produced by recombinant E. coli carrying the α-amylase gene exhibited maximal activity at pH 6.0, identical to α-amylase in the culture filtrate of B. licheniformis, while the temperature profile was somewhat different between the two. Particularly, α-amylase produced from B. lcheniformis is much more thermostable than that from recombinant E. coli. The predominant products resulting from the α-amylase hydrolysis were glucose, maltose and maltotriose for maltotetraose and maltohexaose.

섬유소 분해균을 이용한 건조 청보리 발효사료가 돼지의 In vitro 발효 특성에 미치는 영향

박도연(Do Yeun Park),박중국(Joong Kook Park),조성백(Sung Back Cho),김창현(Chang-Hyun Kim) 한국초지조사료학회 2010 한국초지조사료학회지 Vol.30 No.2

본 연구는 높은 섬유소 분해력을 검증 받은 Aspergillus niger (KCCM 60357)와 Bacillus licheniformis (KCCM 40934)를 단독 및 혼합 배양한 미생물제제로 양돈용 청보리 발효사료를 제조하였을 때 사료 성상변화, in vitro 대장발효 및 전장소화율에 미치는 영향을 평가하였다. 실험 설계는 건조 청보리 (control), A. niger (control + A. niger), B. licheniformis (control + B. licheniformis) 및 Mixture (control + Aspergillus niger + Bacillus licheniformis)구로, 균주를 첨가시킨 발효사료에 따라 네 처리군으로 나누었다. 사료 성분변화는 발효사료의 일반 성분분석을 통해 확인하였으며, 그 결과 A. niger 및 B. licheniformis구에서 조단백질과 조지방 함량이 유의하게 증가하였다 (p<0.05). In vitro 대장발효성상의 변화는 배양 후 24시간에 건물 분해율이 A. niger와 Mixture구에서 각각 55 및 57.42%로 대조구 및 B. licheniformis구와 비교하여 유의하게 높았다 (p<0.05). 가스 발생량은 대조구에서 유의적으로 가장 높았으며, 메탄가스 농도는 모든 처리구가 대조구에 비해 증가되었다. pH는 대조구를 비롯한 모든 처리구가 6.8 로 확인되었으며, 암모니아 질소는 모든 발효사료에서 유의하게 증가되었다. Xylanase 활력은 A. niger구가 유의적으로 높았으며 모든 처리구의 활력이 증가하였다 (p<0.05). 총 VFA 농도는 배양 후 24시간에 B. licheniformis구에서 높은 농도 (12.61mM)를 보여주며 VFA 개선효과를 나타냈다 (p<0.05). In vitro 전장 소화율은 B. licheniformis구에서 49.61%로 대조구의 45.65% 보다 소화율이 유의적으로 증진되었다(p<0.05). 따라서 본 실험에 사용된 미생물을 이용하여 청보리 발효사료를 제조하였을 때 전장 소화율 및 대장발효 환경 개선에 충분한 효과가 있을 것으로 기대된다. The experiment was conducted to observe the effects of dried whole crop barley treated with cellulolytic microorganisms (Aspergillus niger KCCM 60357 and Bacillus licheniformis KCCM 40934) on the chemical composition, in vitro colonic fermentation and whole tract digestibility in swine. Whole crop barley were fermented with no microorganism addition (control), A. niger, B. licheniformis and co-culture of A. niger and B. licheniformis (Mixture) for 3 days at 30℃. In the feed chemical composition, CP contents of whole crop barley treated with A. niger (7.52%) and B. licheniformis (7.77%) were significantly higher than control (6.81%) (p<0.05). The in vitro colonic fermentation of dried whole crop barley fermented with control showed significantly higher CH₄ contents than A. niger, B. licheniformis and Mixture at 18h incubation (p<0.05). Dry matter (DM) digestibilities of A. niger (55%) and Mixture (57.42%) treatments were significantly higher than control (43.74%) (p<0.05). Ammonia-N was significantly increased in A. niger, B. licheniformis and Mixture relative to control at 24 hour incubation (p<0.05). Xylanase activities in A. niger, B. licheniformis and Mixture treatments were significantly higher than control at 24 hour incubation (p<0.05). Concentrations of total VFA were significantly increased in B. licheniformis (12.61 mM) at 24hour incubation (p<0.05). In vitro whole tract digestibility was significantly increased in B. licheniformis (49.61%) compared with the control (45.65%) (p<0.05). In conclusion, whole crop barley treated with cellulolytic microorganisms improved whole tract digestibility and colonic fermentation for swine.

Biosorption of CR Dye in Aqueous Solution by Bacillus licheniformis SRCM 120569 Isolated from Korean Turbid Rice Wine (Makgeolli)

( Jinwon Kim ),( Hee-jong Yang ),( Gwangsu Ha ),( Su-ji Jeong ),( Hee-gun Yang ),( Ho-jin Jeong ),( Se-won Park ),( Do-youn Jeong ) 한국산업식품공학회 2021 산업 식품공학 Vol.25 No.2

The biosorption characteristics of SRCM 120569 biomass on anionic Congo red (CR) dye. SRCM 120569 strain isolated from Korean turbid rice wine (makgeolli) was identified by 16S rRNA sequence analysis as Bacillus licheniformis. Bacillus licheniformis SRCM 120569 (Genbank Accession No. MW819861) showed superior CR dye biosorption capacity in an aqueous solution. Maximum adsorption capacities of 61.2 and 133.1 mg/g were obtained at pH 3.41 and 0.01 g/50 mL dried cell dosage, respectively. The CR dye adsorption properties by the Bacillus licheniformis SRCM120569 strain were characterized by Fourier transform infrared spectroscopy (FT-IR), point of zero charge (pH_pzc) analysis, and phylogenetic analysis. The biosorption isotherm and kinetic models are well described with the Langmuir adsorption isotherm and pseudo-second-order kinetic models.

생활하수에서 분리된 Bacillus licheniformis의 인 제거에 대한 환경적인 인자의 영향

한석순,박상욱,김덕원,박지수,오은지,유진,김덕현,정근욱,Han, Seok-Soon,Park, Sang-Wook,Kim, Deok-Won,Park, Ji-Su,Oh, Eun-Ji,Yoo, Jin,Kim, Deok-Hyeon,Chung, Keun-Yook 한국환경과학회 2021 한국환경과학회지 Vol.30 No.2

This study was initiated to isolate the microorganisms removing phosphorus (P) from domestic sewage and to investigate the effects of environmental factors on the growth and P removal of the isolated bacteria. Microorganisms isolated from the sewage were identified as Chryseobacterium sp., Stenotrophomonas maltophilia, and Bacillus licheniformis. Among them, Bacillus licheniformis was selected as the P removal microorganism. The environmental factors considered in this study included initial phosphorus concentration, temperature, pH, and carbon source. At initial P concentrations of 10, 20, and 30 mg/L, the P removal efficiencies were 100.0%, 84.0%, and 16.5%, respectively. At 20℃, 30℃, and 40℃, the P removal efficiencies were 0%, 75.8%, and 60.6%, respectively. The removal efficiencies of phosphorus according to pH were 1.6%, 91.7%, and 51.1% at pH 5, pH 7, and pH 9, respectively. Using glucose, acetate, and glucose + acetate as carbon sources yielded P removal efficiencies of 80.9%, 33.6%, and 54.1%, respectively. Therefore, the results from the study demonstrated that the P removal efficiencies of Bacillus licheniformis were the highest when the initial P concentration, temperature, pH, and carbon source were 10 mg/L, 30℃, 7, and glucose, respectively.

Bacillus licheniformis WL-12의 cellulase 유전자 클로닝과 발현

윤기홍,Yoon, Ki-Hong 한국미생물학회 2006 미생물학회지 Vol.42 No.4

가정에서 제조된 된장으로부터 cellulase 생산균으로 분리된 고온성 WL-12는 형태적 특성, 생화학적 성질 및 16S rRNA의 염기서열에 근거하여 Bacillus licheniformis로 동정되었다. B. licheniformis WL-12의 cellulase 유전자를 클로닝하여 그 염기서열을 결정한 결과 cellulase 유전자(celA)는 517 아미노산으로 구성된 단백질을 코드하며 1,551 뉴클레오티드로 이루어졌다. 아미노산 잔기배열을 분석한 결과 WL-12의 cellulase는 활성영역과 cellulose 결합영역으로 구성되어 있었으며, glycosyl hydrolase (GH) family 5에 속하는 B. licheniformis, B. subtilis와 B. amyloliquefaciens의 cellulase와 높은 상동성을 보였다. 클론된 celA를 발현용 vector에 도입하여 B. subtilis에서 발현시켜 cellulase 최대생산성이 7.0 units/ml에 이르렀다. A thermophilic bacterium producing the extracellular cellulase was isolated from soybean paste, and the isolate WL-12 has been identified as Bacillus licheniformis on the basis on its 16S rRNA sequence, morphology and biochemical properties. A gene encoding the cellulase of B. licheniformis WL-12 was cloned and its nucleotide sequence was determined. This cellulase gene, designated celA, consisted of 1,551 nucleotides, encoding a polypeptide of 517 amino acid residues. The gene product contained catalytic domain and cellulose binding domain. The deduced amino acid sequence was highly homologous to those of cellulases of B. licheniformis, B. subtilis and B. amytoliquefaciens belonging to the glycosyl hydrolase family 5. When the celA gene was highly expressed using a strong B. subtilis promoter, the extracellular cellulase was produced up to 7.0 units/ml in B. subtilis WB700.

느타리 수확후배지로부터 분리된 고온성 Bacillus licheniformis YJ09의 특성

김혜수,김철환,조수정 한국버섯학회 2016 한국버섯학회지 Vol.14 No.4

In order to isolate thermophilic bacteria with high activity of CMCase and xylanase, spent mushroom substrates was collected from an oyster mushroom cultivation farm in Jinju, Gyeongnam, Korea. Among the isolates, one strain designated as YJ09 was selected by agar diffusion method. The isolate YJ09 was identified as a member of Bacillus licheniformis based on biochemical characteristics using Bacillus ID kit and MicroLog system. Comparative 16S rDNA sequence analysis showed that isolate YJ09 formed a distinct phylogenetic tree within the genus Bacillus and was most closely related to Bacillus licheniformis with sequence similarity of 98.9%. Based on its physiological properties, biochemical characteristics and phylogenetic distinctiveness, the isolate YJ09 was classified as Bacillus licheniformis. The CMCase and xylanase activity of B. licheniformis YJ09 was slightly increased corresponding to the bacterial population from exponential phase to stationary phase in the growth curve of B. licheniformis YJ09.

Heterologous Gene Expression of aprE2 Encoding a 29 kDa Fibrinolytic Enzyme from Bacillus subtilis in Bacillus licheniformis ATCC 10716

Gun-Hee Kwon,Woo-Ju Jeong,Ae Ran Lee,Jae-Yong Park,Jaeho Cha,Young-Sun Song,Jeong Hwan Kim 한국식품과학회 2008 Food Science and Biotechnology Vol.17 No.6

The aprE2 gene from Bacillus subtilis CH3-5 was expressed in Bacillus licheniformis ATCC 10716 using a Bacillus-Escherichai coli shuttle vector, pHY300PLK. The fibrinolytic activity of transformant (TF) increased significantly compared to B. licheniformis 10716 control cell. During the 100 hr incubation in Luria-Bertaini broth at 37℃, fibrinolytic activity of B. licheniformis TF increased rapidly at the late growth stage, after 52 hr of incubation, which was confirmed by zymography using a fibrin gel. pHY3-5 was stably maintained in B. licheniformis without tetracycline (Tc) in the media, 60.9% of cells still maintained pHY3-5 after 100 hr of cultivation.

청국장 유래 Bacillus licheniformis의 ${\beta}$-Galactosidase 특성

윤기홍,Yoon, Ki-Hong 한국미생물·생명공학회 2012 한국미생물·생명공학회지 Vol.40 No.1

가정에서 제조된 청국장으로부터 lactose를 glucose와 galactose로 가수분해하는 ${\beta}$-galactosidase의 생산균을 분리하였다. 분리균 YB-1105는 형태적 특성, 생화학적 성질 및 16S rRNA 유전자 염기서열에 근거하여 Bacillus licheniformis로 확인되었다. B. licheniformis YB-1105의 배양상등액과 균체파쇄액에서 모두 ${\beta}$-galactosidase 활성이 관찰되었으며 이들은 모두 pH 6.5와 $50^{\circ}C$의 반응조건에서 paranitrophenyl-${\beta}$-D-galactopyranoside의 가수분해 활성이 최대로 나타났다. 그러나 균체파쇄상등액에 비해 배양상등액의 ${\beta}$-galactosidase 활성은 산성 pH와 고온에서 크게 영향을 받았다. 한편 두 분획의 가수분해 활성은 낮은 농도의 galactose에 의해도 급격하게 저해되었으나, glucose와 mannose는 고농도에 의해서는 약하게 저해를 받는 것으로 확인되었다. A bacterial strain was isolated from homemade Cheongkookjang as a producer of the ${\beta}$-galactosidase, capable of hydrolyzing lactose to liberate galactose and glucose residues. The isolate YB-1105 has been identified as Bacillus licheniformis on the basis of its 16S rDNA sequence, morphology and biochemical properties. ${\beta}$-Galactosidase activity was detected in both the culture supernatant and the cell extract of B. licheniformis YB-1105. The enzymes of both fractions demonstrated maximum activity for hydrolysis of para-nitrophenyl-${\beta}$-D-galactopyranoside (pNP-${\beta}Gal$) under identical reaction conditions of pH 6.5 and $50^{\circ}C$. However, ${\beta}$-galactosidase activity from the culture filtrate was affected more than that from the cell free extract at acidic pHs and high temperatures. The hydrolyzing activity of both ${\beta}$-galactosidases for pNP-${\beta}Gal$ was dramatically decreased by the addition of low concentrations of galactose, but was only marginally decreased by high concentrations of glucose or mannose.

The difference in <i>in vivo</i> sensitivity between <i>Bacillus licheniformis</i> PerR and <i>Bacillus subtilis</i> PerR is due to the different cellular environments

Kim, Jung-Hoon,Won, Young-Bin,Ji, Chang-Jun,Yang, Yoon-Mo,Ryu, Su-Hyun,Ju, Shin-Yeong,Kwon, Yumi,Lee, Yeh-Eun,Lee, Jin-Won Elsevier 2017 Biochemical and biophysical research communication Vol.484 No.1

<P><B>Abstract</B></P> <P>PerR, a member of Fur family of metal-dependent regulators, is a major peroxide sensor in many Gram positive bacteria, and controls the expression of genes involved in peroxide resistance. <I>Bacillus licheniformis</I>, a close relative to the well-studied model organism <I>Bacillus subtilis</I>, contains three PerR-like proteins (PerR<SUB>BL</SUB>, PerR2 and PerR3) in addition to Fur and Zur. In the present study, we characterized the role of PerR<SUB>BL</SUB> in <I>B. licheniformis</I>. <I>In vitro</I> and <I>in vivo</I> studies indicate that PerR<SUB>BL</SUB>, like PerR<SUB>BS</SUB>, uses either Fe<SUP>2+</SUP> or Mn<SUP>2+</SUP> as a corepressor and only the Fe<SUP>2+</SUP>-bound form of PerR<SUB>BL</SUB> senses low levels of H<SUB>2</SUB>O<SUB>2</SUB> by iron-mediated histidine oxidation. Interestingly, regardless of the difference in H<SUB>2</SUB>O<SUB>2</SUB> sensitivity, if any, between PerR<SUB>BL</SUB> and PerR<SUB>BS</SUB>, <I>B. licheniformis</I> expressing PerR<SUB>BL</SUB> or PerR<SUB>BS</SUB> could sense lower levels of H<SUB>2</SUB>O<SUB>2</SUB> and was more sensitive to H<SUB>2</SUB>O<SUB>2</SUB> than <I>B. subtilis</I> expressing PerR<SUB>BL</SUB> or PerR<SUB>BS</SUB>. This result suggests that the differences in cellular milieu between <I>B. subtilis</I> and <I>B. licheniformis</I>, rather than the intrinsic differences in PerR<SUB>BS</SUB> and PerR<SUB>BL</SUB> <I>per se</I>, affect the H<SUB>2</SUB>O<SUB>2</SUB> sensing ability of PerR inside the cell and the H<SUB>2</SUB>O<SUB>2</SUB> resistance of cell. In contrast, <I>B. licheniformis</I> and <I>B. subtilis</I> expressing <I>Staphylococcus aureus</I> PerR (PerR<SUB>SA</SUB>), which is more sensitive to H<SUB>2</SUB>O<SUB>2</SUB> than PerR<SUB>BL</SUB> and PerR<SUB>BS</SUB>, were more resistant to H<SUB>2</SUB>O<SUB>2</SUB> than those expressing either PerR<SUB>BL</SUB> or PerR<SUB>BS</SUB>. This result indicates that the sufficient difference in H<SUB>2</SUB>O<SUB>2</SUB> susceptibility of PerR proteins can override the difference in cellular environment and affect the resistance of cell to H<SUB>2</SUB>O<SUB>2</SUB>.</P> <P><B>Highlights</B></P> <P> <UL> <LI> <I>Bacillus licheniformis</I> PerR senses H<SUB>2</SUB>O<SUB>2</SUB> by Fe-mediated histidine oxidation. </LI> <LI> The <I>in vivo</I> H<SUB>2</SUB>O<SUB>2</SUB>-sensing ability of PerR can be modulated by cellular environments. </LI> <LI> The H<SUB>2</SUB>O<SUB>2</SUB>-sensitivity of PerR can affect the resistance of cells to H<SUB>2</SUB>O<SUB>2</SUB>. </LI> </UL> </P>

된장에서 분리된 Bacillus licheniformis의 ${\beta}$-galactosidase 생산성과 효소특성

진현경,윤기홍,Jin, Hyun Kyung,Yoon, Ki-Hong 한국미생물·생명공학회 2014 한국미생물·생명공학회지 Vol.42 No.4

가정에서 제조된 된장으로부터 lactose를 glucose와 galactose로 가수분해하는 균체외 ${\beta}$-galactosidase의 생산균이 분리되었다. 분리균 YB-1414는 형태적 특성, 생화학적 성질 및 16S rRNA 유전자 염기서열에 근거하여 Bacillus licheniformis로 확인되었다. 탄소원과 질소원으로 밀기울 (1%)과 yeast extract (2.5%)를 사용하였을 때 B. licheniformis YB-1414의 ${\beta}$-galactosidase 생산성이 최대 6.2 U/ml에 이르렀다. 특히 밀기울의 불용성 성분이 수용성 성분보다 ${\beta}$-galactosidase 생산성을 더 증가 시키는 것으로 확인되었다. ${\beta}$-galactosidase의 para-nitrophenyl-${\beta}$-D-galactopyranoside 가수분해 활성은 pH 6.0과 $55-60^{\circ}C$에서 가장 높았으며, 낮은 농도의 galactose에 의해서도 크게 저해를 받았다. 그러나 glucose에 의해서는 ${\beta}$-galactosidase의 가수분해 활성이 약하게 저해를 받으며 400 mM glucose가 존재하여도 최대 활성의 85%에 해당하는 가수분해 활성을 보였다. A bacterial strain was isolated from homemade doenjang (Korean fermented soybean paste) as a producer of the extracellular ${\beta}$-galactosidase, capable of hydrolyzing lactose to liberate galactose and glucose residues. The isolate YB-1414 has been identified as Bacillus licheniformis on the basis of its 16S rDNA sequence, morphology and biochemical properties. The production of ${\beta}$-galactosidase by B. licheniformis YB-1414 reached maximum levels of 6.2 U/ml in culture medium containing wheat bran (1%) and yeast extract (2.5%) as carbon and nitrogen sources, respectively. Particularly, the insoluble fraction was more effective for ${\beta}$-galactosidase production than the soluble extract of wheat bran. The enzyme exhibited maximum activity for hydrolysis of para-nitrophenyl-${\beta}$-D-galactopyranoside (pNP-${\beta}Gal$) under reaction conditions of pH 6.0 and $55-60^{\circ}C$. Its hydrolyzing activity for pNP-${\beta}Gal$ was drastically decreased by the addition of low concentrations of galactose, but only slightly decreased by glucose, with 85% of maximal activity in the presence of 400 mM glucose.